Abstract Nonhomologous end joining (NHEJ) is a major pathway for resolving chromosome double strand breaks. NHEJ deficiency results in a wide variety of phenotypes including premature cellular senescence, immunodeficiency, defective neurogenesis, dwarfism, accelerated aging, and cancer predisposition. Notably, NHEJ is mostly dispensable in resolving the clean chromosome breaks used in many experimental models. Why, then, are the phenotypes of NHEJ deficiency so severe? We will address here if NHEJ's pivotal role in maintaining genome stability is at least in part due to a unique ability to flexibly cope with the complex end structures expected in biologically relevant contexts. Of particular interest to this proposal, broken ends generated by ionizing radiation, radiomimetic drugs, or chronic oxidative stress possess associated damage to flanking DNA, including oxidized and missing bases. The ability of ionizing radiation and radiomimetic drugs to introduce such complex damage is a major factor in the biological impact of these agents, and thus critical in the effectiveness of these agents in tumor therapy. In Aim 1, we will determine how associated radiomimetic damage effects how NHEJ joins ends, both in vitro and in cells. Ku is a key factor in repair of breaks by NHEJ. In Aim 2 we will address the extent to which Ku's recently described ability to excise damaged nucleotides from ends contributes to its role in NHEJ. In Aim 3 we will determine how NHEJ regulates the fidelity of the ligation step, and what impact regulation of fidelity has on resolution of complex end structures.